The US Energy Independence and Security Act (EISA) of 2007 states that transportation fuel introduced into commerce in the US (annual average) contain at least 12.95 billion gallons of renewable fuels by the year 2010. Ethanol is the most prevalent renewable fuel, with the US producing over 6 billion gallons in 2007 (Peterson and Ingram, 2008, Ann. N.Y. Acad. Sci., 1125:363-372) and 9 billion gallons in 2009. Currently, the majority of ethanol is produced from corn; however, limited supply will force ethanol production from other sources of biomass, of which the US produces over a billion tons annually—enough to produce 80 billion gallons of renewable fuel (Gray et al., 2006, Bioethanol. Curr. Opin. Chem. Biol., 10:141). Moreover, use of waste biomass for fuel production positively affects greenhouse gases and carbon debt without causing land-use change (Fargione et al., 2008, Science, 319:1235-1237, Searchinger et al., 2008, Science, 319:1238-1240). The EISA of 2007 also requires that ethanol, and other liquid transportation fuels such as butanol and biomass-based diesel, derived from any of the following: cellulose, hemicellulose, lignin, sugar, starch (other than corn starch), waste material and residues, be incorporated into our 2010 fuel supply at 0.95 billion gallons. By the year 2015 over 5 billion gallons of advanced renewable fuel from biomass other than corn starch are required to be available for use in our transportation sector.
Unlike corn grain, where the major component is starch, other sources of biomass are composed of 40-50% cellulose, 25-35% hemicellulose, and 15-20% lignin (Gray et al., 2006, Bioethanol. Curr. Opin. Chem. Biol., 10:141, Doran-Peterson et al., 2008, The Plant J., 54:582-592). The highly complex biomass structure has necessitated development of many processes for fuel ethanol conversions from substrates containing lignocellulose, which can include thermochemical and/or mechanical pretreatment to allow enzymatic access, enzymatic degradation to reduce substrates to fermentable sugars, and finally fermentation of those sugars by microorganisms. Commercially available enzyme mixtures are usually culture supernatants from fungi, and sometimes bacteria, containing a complex of enzyme activities. In order to efficiently degrade cellulose several major classes of enzymes are required, such as endo-β-1,4-glucanases (endocellulase, Cx-cellulase; EC 3.2.1.4) which cleave internal β-1,4-glycosidic bonds generating oligosaccharides; exo-β-1,4-glucanases (exocellulase, cellobiohydrolaseC1-cellulase; EC 3.2.1.91) which cleave the non-reducing end to release a dimer of glucose called cellobiose; and β-glucosidase (cellobiase, EC 3.2.1.21) which cleaves cellobiose into monomeric glucose molecules (Whitaker, 1994, Principles of Enzymology for the Food Sciences, 2nd Ed. Marcel Dekker, New York, Gilkes et al., 1991, Microbiol Rev., 55:303-315, Henrissat et al., 1989, Gene, 81(1):83-95, Béguin et al., 1994, FEMS Microbiol Rev., 1994 13(1):25-58). Many commercial preparations are deficient in cellobiase, and when this dissacharide accumulates it can inhibit further enzyme deconstruction of the cellulose microfibrils.
In some biomass types, such as sugar beet pulp and citrus peel, pectin can also compose a significant portion of the lignocellulose structure and functions as a matrix to hold cellulose and hemicellulose fibers. The pectin backbone can consist of a homopolymer of α-1,4-D-galacturonic acid (homogalacturonan) or repeats of the disaccharide α-1,2-L-rhamnose-α-1,4-D-galacturonic acid (rhamnogalacturonan-I), and, typically, 70% to 80% of galacturonic acid residues are methylated. Homogalacturonan can be substituted with xylose or apiose, while rhamnogalacturonan-I is often substituted with galactose, arabinose, or galactan (Willats et al., 2001, Plant Mol. Biol., 47:9-27, Ridley et al., 2001, Phytochemistry, 57:929-967).
The degradation of pectin requires both methylesterases and depolymerases. Pectin methylesterases are responsible for the hydrolysis of methylester linkages from the polygalacturonic acid backbone (Whitaker, 1984, Enzyme Microbial Technol., 6:341-347). Pectin depolymerases act upon the polygalacturonate backbone and belong to one of two families: polygalacturonases or lyases. Polygalacturonases are responsible for the hydrolytic cleavage of the polygalacturonate chain, while lyases cleave by β-elimination giving a Δ4,5-unsaturated product (Jayani et al., 2005, Process Biochem., 40:2931-2944, Sakai et al., 1993, Adv. Appl. Microbiol., 39:231-294). There are two types of lyases: pectate lyases, which cleave unesterified polygalacturonate, or pectate; and pectin lyases, which cleave methyl esterified pectin. Pectate lyases have been classified into families based on amino acid similarity, which in turn suggests structural features (Coutinho and Henrissat, 1999, In: Gilbert et al. (Eds.) Recent Advances in Carbohydrate Bioengineering. Cambridge, The Royal Society of Chemistry).
Once the lignocellulosic biomass is degraded into fermentable sugars, many different types of sugars, including pentose and acidic sugars are liberated for metabolism to a product(s) (Doran-Peterson et al., 2008, The Plant J., 54:582-592). Most ethanol fermentations in the U.S. today use the yeast Saccharomyces cerevisiae to convert starch glucose into ethanol and CO2; however, lignocellulosic biomass contains many sugars that S. cerevisiae is unable to ferment (Peterson and Ingram, 2008, Ann. N.Y. Acad. Sci., 1125:363-372). Thus, Escherichia coli, which is capable of using these hexoses and pentoses, was engineered as a biocatalyst for ethanol production by integration of the pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) genes from Zymomonas mobilis into the chromosome of E. coli to generate strain K011 (Ohta et al., 1991, Appl. Environ. Microbiol., 57:893-900).